Journal: NPJ Breast Cancer

Article Title: Synergistic targeting of BRCA1 mutated breast cancers with PARP and CDK2 inhibition

doi: 10.1038/s41523-021-00312-x

Figure Lengend Snippet: a HCC1937 cells treated with fadraciclib (CYC065) or vehicle for 5 days were analyzed by alkaline Comet assay, 90–300 tails quantitated/treatment in triplicate experiments. Data analyzed by one-way ANOVA; ** p < 0.01, *** p < 0.001. Representative images shown, scale bar is 50 μm. b HCC1937 cells treated with CVT313 or vehicle for 5 days were analyzed by alkaline Comet assay, 100–275 tails quantitated/treatment in triplicate experiments. Data analyzed by one-way ANOVA; *** p < 0.001, **** p < 0.0001. Representative images shown, scale bar is 50 μm. c HCC1937 cells treated with CDK2 siRNA or Non-targeting Pool siRNA for 72 h were analyzed by alkaline Comet assay, 75–250 tails quantitated/treatment. Triplicate experiments analyzed by one-way ANOVA; * p < 0.05, ** p < 0.01, **** p < 0.0001. Representative images shown, scale bar is 50 μm. Inset are western blots of matched lysates for CDK2 and GAPDH. d HCC1937 cells treated with CDK9 siRNA or Non-targeting Pool siRNA for 72 h were analyzed by alkaline Comet assay, 130–300 tails quantitated/treatment. Triplicate experiments analyzed by one-way ANOVA; * p < 0.05. Representative images shown, scale bar is 50 μm. Inset are western blots of matched lysates for CDK9 and GAPDH. e HCC1937 cells were treated with doses of fadraciclib and rucaparib for 5 days, and viability measured by Alamar Blue. Synergy analysis was performed by BLISS, where blue indicates synergy, and red indicates antagonism. Data are pooled from 5 replicates. f MDA-MB-436 cells were treated with doses of fadraciclib and rucaparib for 5 days, and viability measured by Alamar Blue. Synergy analysis was performed by BLISS, where blue indicates synergy, and red indicates antagonism. Data are pooled from 3 replicates. g BT-20 cells were treated with doses of fadraciclib and rucaparib for 5 days, and viability measured by Alamar Blue. Synergy analysis was performed by BLISS, where blue indicates synergy, and red indicates antagonism. Data are pooled from 8 replicates. h MDA-MB-468 cells were treated with doses of fadraciclib and rucaparib for 5 days, and viability measured by Alamar Blue. Synergy analysis was performed by BLISS, where blue indicates synergy, and red indicates antagonism. Data are pooled from 3 replicates.

Article Snippet: HCC1937 cells were seeded in a 6 well plate and treated with fadraciclib (Cyclacel) or CVT313 (Thermofisher) at the calculated IC 5 , IC 20 or IC 50 dose for 5 days, or treated with 10, 20, or 50 nM CDK2 siRNA or CDK9 siRNA, or 50 nM non targeting siRNA for 72 h. Slides were imaged with a fluorescence microscope (Leica DM5500) and analyzed with ImageJ OpenComet software (v1.3.1, ).

Techniques: Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: NPJ Breast Cancer

Article Title: Synergistic targeting of BRCA1 mutated breast cancers with PARP and CDK2 inhibition

doi: 10.1038/s41523-021-00312-x

Figure Lengend Snippet: a Schematic for drug administration to BLBC PDX models. b BLBC BRCA1 R1443* PDX model was treated with vehicle (gray, n = 10), fadraciclib (CYC065) 25 mg/kg (red, n = 9), olaparib 50 mg/kg (blue, n = 8) or the combination of fadraciclib and olaparib (purple, n = 9). Tumor volume was measured with calipers for 8 weeks. Data analyzed by repeated measures one-way ANOVA; * p < 0.05, ** p < 0.01, **** p < 0.0001. c Growth kinetics of individual BRCA1 R1443* PDX tumors with therapy. d Kaplan–Meier survival curves of ( b ), statistical differences between curves estimated by the Logrank (Mantel–Cox) test; * p < 0.05, **** p < 0.0001. e BLBC BRCA1 2080delA PDX model was treated with vehicle (gray, n = 3), fadraciclib 25 mg/kg (red, n = 5), olaparib 50 mg/kg (blue, n = 5) or the combination of fadraciclib and olaparib (purple, n = 4). Tumor volume was measured with calipers for 7 weeks. f Growth kinetics of individual BRCA1 2080delA PDX tumors with therapy. g The change in tumor volume in both BRCA1 R1443* and BRCA1 2080delA PDX models between start of treatment and ethical endpoint for olaparib and combination treated cohorts. Endpoint for BRCA1 R1443* PDX was 60 days. Endpoint for BRCA1 2080delA PDX was 33 days. Data analyzed by two-tailed paired t -test; * p < 0.05, *** p < 0.001. h BLBC HCI-002 ( BRCA1 WT, BRCA2 WT) PDX was treated with vehicle (gray, n = 6), fadraciclib 25 mg/kg (red, n = 7), olaparib 50 mg/kg (blue, n = 8) or the combination of fadraciclib and olaparib (purple, n = 6). Tumor volume was measured with calipers for 5 weeks, WT = wildtype. i Growth kinetics of individual HCI-002 ( BRCA1 WT, BRCA2 WT) PDX tumors with therapy, WT = wildtype. j Kaplan–Meier survival curves of ( h ), statistical differences between curves estimated by the Logrank (Mantel–Cox) test; N.S. = not significant. k γ-H2AX IHC of vehicle and combination fadraciclib and olaparib treated tumors of the BRCA1 R1443* PDX models. Scale bar is 200 µm . l Quantitation of high intensity γ-H2AX foci in vehicle and combination treated tumors, analyzed by two-sided t -test. m Western blots for MCL-1 and β-Actin in lysates from vehicle ( n = 3), fadraciclib ( n = 4), olaparib ( n = 4) and combination fadraciclib and olaparib ( n = 4) treatment. All samples derive from the experiment shown in ( b ) and western blots were processed in parallel.

Article Snippet: HCC1937 cells were seeded in a 6 well plate and treated with fadraciclib (Cyclacel) or CVT313 (Thermofisher) at the calculated IC 5 , IC 20 or IC 50 dose for 5 days, or treated with 10, 20, or 50 nM CDK2 siRNA or CDK9 siRNA, or 50 nM non targeting siRNA for 72 h. Slides were imaged with a fluorescence microscope (Leica DM5500) and analyzed with ImageJ OpenComet software (v1.3.1, ).

Techniques: Two Tailed Test, Quantitation Assay, Western Blot